Figure 1.
Figure 1. Methionine stimulation induces dephosphorylation of Art1. (A) Yeast cells expressing Mup1-GFP (green) and Vph1-mCherry (red; localized to the limiting membrane of the vacuole) were grown to mid-log phase and imaged by fluorescence deconvolution microscopy (untreated) or stimulated with methionine for the indicated amount of time and then imaged. (B) Yeast cells (WT or Δart1) expressing Mup1-pHluorin were cultured to mid-log phase, and flow cytometry was used to measure pHluorin-positive cells at the indicated time points following methionine stimulation (+ met.) or an untreated control culture. Sample data of the Mup1-pHluorin–trafficking analysis by microscopy and flow cytometry are provided in Fig. S1 A. Error bars indicate standard deviation from multiple biological replicate experiments (n = 3). (C) Yeast cells expressing Art1-GFP (green) and Mup1-MARS (red) were grown to mid-log phase and imaged by fluorescence deconvolution microscopy (untreated) or stimulated with methionine for 3 min and then imaged. (D) Pearson’s correlation coefficients were measured for yeast cells (n > 25 cells each for untreated or methionine stimulation conditions) expressing Art1-GFP and Mup1-MARS-RFP (as shown in C). Error bars indicate standard deviation for the set, and significance was determined by an unpaired t test. (E) SILAC-based quantification of phosphorylation events was performed (according to the scheme shown in Fig. S1 B) using endogenous Art1-FLAG as bait. A schematic representation of Art1 (top) is shown with domains color-coded to indicate location of phosphopeptides detected and quantified in this analysis (bottom graph). Normalized H:L ratios were measured and averaged over multiple biological replicates (n = 3). Error bars indicate standard deviation of measurements on peptides from three experiments (n = 3), except for S104 and S113, which were only resolved and quantified in two replicate experiments. (F) Complementation analysis of Δart1 mutant yeast cells using the Mup1-pHluorin–trafficking assay. For all Mup1-pHluorin–trafficking assays plots represents average of multiple biological replicate experiments (n ≥ 4), and error bars indicate standard deviation. For the final five time points of each time course, statistical significance was calculated using an unpaired t test (P < 0.005).

Methionine stimulation induces dephosphorylation of Art1. (A) Yeast cells expressing Mup1-GFP (green) and Vph1-mCherry (red; localized to the limiting membrane of the vacuole) were grown to mid-log phase and imaged by fluorescence deconvolution microscopy (untreated) or stimulated with methionine for the indicated amount of time and then imaged. (B) Yeast cells (WT or Δart1) expressing Mup1-pHluorin were cultured to mid-log phase, and flow cytometry was used to measure pHluorin-positive cells at the indicated time points following methionine stimulation (+ met.) or an untreated control culture. Sample data of the Mup1-pHluorin–trafficking analysis by microscopy and flow cytometry are provided in Fig. S1 A. Error bars indicate standard deviation from multiple biological replicate experiments (n = 3). (C) Yeast cells expressing Art1-GFP (green) and Mup1-MARS (red) were grown to mid-log phase and imaged by fluorescence deconvolution microscopy (untreated) or stimulated with methionine for 3 min and then imaged. (D) Pearson’s correlation coefficients were measured for yeast cells (n > 25 cells each for untreated or methionine stimulation conditions) expressing Art1-GFP and Mup1-MARS-RFP (as shown in C). Error bars indicate standard deviation for the set, and significance was determined by an unpaired t test. (E) SILAC-based quantification of phosphorylation events was performed (according to the scheme shown in Fig. S1 B) using endogenous Art1-FLAG as bait. A schematic representation of Art1 (top) is shown with domains color-coded to indicate location of phosphopeptides detected and quantified in this analysis (bottom graph). Normalized H:L ratios were measured and averaged over multiple biological replicates (n = 3). Error bars indicate standard deviation of measurements on peptides from three experiments (n = 3), except for S104 and S113, which were only resolved and quantified in two replicate experiments. (F) Complementation analysis of Δart1 mutant yeast cells using the Mup1-pHluorin–trafficking assay. For all Mup1-pHluorin–trafficking assays plots represents average of multiple biological replicate experiments (n ≥ 4), and error bars indicate standard deviation. For the final five time points of each time course, statistical significance was calculated using an unpaired t test (P < 0.005).

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